Complete Chloroplast Genome Sequence And Development Of Chloroplast Polymorphic Molecular Markers In Leymus Chinensis

The chloroplast genome is very important for the study on chloroplast gene engineering,cultiving high quality varieties,phylogeography,and phylogenetic of low taxon species.We sequenced and acquired the complete chloroplast genome of Leymus chinensis which was one family of Triticeae after performed assembling,gene functional annotation.We carried on genome characteristic analysis,phylogenetic analysis,and compared the relative species genome.Based on the chloroplast genome,we also developed the polymorphism of non-coding fragment sequences and SSR(Simple Sequence Repeat)molecular markers.The chloroplast genome sequencing was first in Leymus Hochst.It provided the basic information for future reaearch on forage chloroplast genome,gene engineering and phylogenetic,and builded a foundation for further molecular marker chloroplast genome.We acquired and analyzed the complete chloroplast genome of Leymus chinensis firstly.The whole genome was 136 783 bp long,and had a typical quadripartite structure of large(LSC,80 946 bp)and small(SSC,12 715 bp)single-copy regions separated by a pair of inverted repeats(IRa,IRb,21 561 bp).The genome contained 131 unique genes,including 91 protein-coding genes,32 transfer RNAs,and 8 ribosomal RNAs.The 91 protein-coding genes encoded 22 917 codes.A total of 26 SSR sites and 49 tandem repeats were detected in the genome.The SSR analysis revealed that most SSRs were AT-rich.The coding regions were found to be more conservative than non-coding regions in the complete chloroplast genome of Leymus chinensis by the comparison of chloroplast genome.Most of the variation happened in the non-coding regions.The IR contraction was more conservative than the LSC and SSC.The IR contraction and expansion events showed different location of rps19 and ndhH genes.The phylogenetic analysis demonstrated that Leymus chinensis was closely related to Triticum timopheevii and Agropyron cristatum.We analysed the polymorphism of 12 cpDNA non-coding fragment sequences and found that the polymorphism sites of ndhF-rpl32?trnL-trnF?trnC-ycf6?aptl-aptH sequences were rich.There were 15 haplotypes identified based on combined sequences in 9 different populations of Leymus chinensis.The haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.928 and 0.00101.The genetic differentiation coefficient Fst was 0.58884 with the low gene flow(Nm=0.17).Tajima's D and Fu,s Fs values of neutrality tests were negative(Tajima's D=-1.0854;Fu's Fs=-5.301).Analysis of Molecular Variance(AMOVA)showed that most of genetic variation distributed among populations(65%).The Mantel test showed a significantly positive correlation between genetic distance and geographical distance(R=0.449,P<0.05).The phylogenetic analysis and network for cpDNA haplotypes based on combined sequence showed that the relationship between H2 and H10(Pop6 and Pop8)had the close relationship.There were 8 SSR sites with polymorphism obtained from 26 cpSSR sites.The genetic diversity of Nei's(h)was 0.2024,and Shannon's information index(I)was 0.3095.The average genetic diversity Nei's index of 9 different populations(h)was 0.0587,and the average Shannon's information index(I)was 0.0865.On the whole,the SSR polymorphism was poor and genetic diversity was low.Both phylogenetic and genetic structural analysis showed that the Pop6 and Pop8 had the closest relationship.These results were similar to the non-coding sequence.